The present invention relates to a novel compound and method for preparation of polyethylene glycol (PEG) adducts of biomolecules, and particularly to PEG adducts of proteins and peptides.
Attachment of large macromolecules such as polyethylene glycol (PEG) to biomolecules such as proteins or peptides via chemical attachment is desired for modification of the properties of the proteins or peptides. Linking to PEG is referred to in the art as xe2x80x9cpegylationxe2x80x9d. Biomolecules circulating in the blood outside of a cell are subject to clearance, and can move through blood vessel walls (extravascularization). Attachment of relatively small biomolecules to large macromolecules can reduce extravascularization, and can enhance the in-vivo circulation half-life of the biomolecule.
Increasing half-life of the biomolecule in circulation is particularly important when the biomolecule is intended for therapeutic use. Pegylation of certain biomolecules reduces kidney clearance and spurious enzymatic degradation and immune system recognition. In xe2x80x9cArtificial Bloodxe2x80x9d, Science, 295:1002, 1004-1005 (Feb. 8, 2002), Jerry E. Squires cites literature reports that conjugation of hemoglobin to macromolecules such as dextran, polyethylene glycol or polyoxyethylene retards the rate at which cell-free hemoglobin is cleared from blood circulation, extending intravascular dwell time up to 48 hours. The alteration of the effective solution volume of the hemoglobin through linkage to a macromolecule alters the colligative properties of cell-free hemoglobin, including osmotic pressure that appears to have a significant effect on blood pressure.
Polyethylene glycol is approved by the U.S. Food and Drug Administration for internal and topical use due to its low toxicity. Additional utilities and features of PEG-biomolecule conjugates are described in Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, J. M. Harris, ed. Plenum, NY, 1992 and Polyethylene Glycol Chemistry and Biological Applications, J. M. Harris and S. Zalipsky, eds., ACS, Washington, 1997.
A requisite for preparation of polyethylene glycol conjugates of biomolecules is a suitably activated PEG molecule that under proper conditions reacts with a target biomolecule in an efficient, predictable manner such that the native activity of the target biomolecule is not adversely affected to a significant degree. For sensitive target biomolecules such as proteins and peptides, reactions to form PEG conjugates are best conducted in aqueous, buffered systems in order to avoid denaturation and concomitant loss of biological activity.
Most, if not all, activated polyethylene glycol compounds described in the patent and scientific literature and intended for conjugation to biomolecules such as proteins react with water in addition to the target biomolecule. See, for example, Scheme I below. Typically, the conundrum of derivatizing a target biomolecule in an aqueous medium with a water-sensitive; activated, polyethylene glycol reagent is partially solved by employing a large excess (5-10 fold) of reagent, while maintaining rigorous control of pH and temperature. An objective of any pegylation procedure is to produce pegylated biomolecules with stable, enhanced physiological properties in a predictable and reproducible manner on a meaningful, economical scale.
Scheme 1, below represents a reaction between an activated pegylation reagent of the art and a biomolecule reactant having acidic hydrogens (hydrido groups). The scheme shows the formation of hydrolysis product, pegylated biomolecule and protonated leaving group (HA). As illustrated in Scheme 1, activated pegylation reagents described in the current art often involve production of a leaving group (HA in Scheme 1 below) in addition to any hydrolyzed PEG reagent (shown as PEG-OH). The production of a leaving group presents an additional workup and purification problem during the isolation of the PEG-modified target molecule. The extent of the isolation/purification problem is influenced by the magnitude of the excess reagent employed in order to achieve the desired level of PEG modification which, in turn, is a function of hydrolysis rate of the reagent in water and the coupling rate onto a residue of the target molecule (typically xe2x80x94NH2 or xe2x80x94SH). 
Typically, activated polyethylene glycol reagents described and used in the current art are acylating reagents directed toward primary amine residues (xe2x80x94NH2) in the target biomolecule. A common type of PEG acylating reagent is the so-called xe2x80x9cactive esterxe2x80x9d derivative of PEG. Active ester PEGs of the N-hydroxysuccinimide (NHS), hydroxybenztriazole (HBT), imidazole (IM)and p-nitrophenol (PNP) have been described and are commercially available. (See Shearwater Corporation, Catalog 2001, Huntsville, Ala. 35801, www.shearwatercorp.com).
Reagents of the type
mPEG-Rxe2x80x94C(O)xe2x80x94OX,
where R=(O)Cxe2x80x94(CH2)y or (CH2)y or (xe2x80x94Oxe2x80x94),
y=zero through 4, and
X=NHS, HBT, IM or PNP
exhibit half-lives of hydrolysis of 1 minute (or less) to approximately 24 minutes at pH=8 and 25xc2x0 C. Further, as half-life goes up, reactivity goes down. Recommended excesses of PEG acylating reagent vary from equal mass to 10-fold mass relative to target molecule (Shearwater Catalog 2001 p.12). Depending on the molecular weight of the target biomolecule, this recommended mass excess can be greater than a 100-1000 molar excess.
Incorporation of macromolecules such as PEG into biomolecules by using currently-available PEG acylating reagents is a demonstrably inefficient process. Problems associated with the acylating PEG reagents of the current art are exacerbated on a large scale. Variables affecting efficiency and reproducibility of pegylation procedures based on current art acylating reagents include: half-life (t1/2) of hydrolysis, pH value, temperature, time, mixing rate, nature and toxicity of leaving group, ease of product purification from leaving groups and hydrolyzed reagent, as well as the rate and extent of reactivity of the reagent toward the target biomolecule.
The rapid hydrolysis rates of acylating PEG reagents employed by the standard art preclude practical application of multi-functional, crosslinking pegylation reagents of the following type, where A is a leaving group. 
A multifunctional, activated PEG reagent as shown above is useful for establishing intramolecular cross-links within protein molecules for the purpose of mapping sub-unit geographies and for stabilization of protein configurations and activities. Rapid hydrolysis rates of standard art PEG reagents, used at large molar excess favor a preponderance of xe2x80x9cone on hitsxe2x80x9d, where one carboxyl end of the bifunctional molecule links to the target protein but the other end hydrolyzes to a carboxylic acid instead of also linking to the protein, and forming stabilizing cross-links.
In a significant advance, workers at the Albert Einstein College of Medicine of Yeshiva University N.Y., University of Iowa and BioAffinity Systems describe a novel approach for activating PEG for attachment to biomolecules that largely circumvents problems associated with hydrolytically unstable reagents. As disclosed in Acharya et al. U.S. Pat. No. 5,585,484, U.S. Pat. No. 5,750,725, U.S. Pat. No. 6,017,943, entitled xe2x80x9cHemoglobin Crosslinkersxe2x80x9d, and Belur N. Manjula, et al., J. Biol. Chem., 275(8):5527-5534 (2000), a maleimide-activated PEG reagent is employed to form stable thioether bonds with an indigenous or added sulfhydryl moiety in the biomolecule. The maleimide function reacts rapidly with xe2x80x94SH groups without significant hydrolysis at pH 6.5-7.0 and without the production of a leaving group as is shown in the reaction below, wherein Rxe2x80x94SH is a sulfhydryl-containing biomolecule. 
Biomolecules such as a protein having a paucity of xe2x80x94SH groups must first be reacted with a thiolating reagent such as 2-iminothiolane (or the like) to convert native xe2x80x94NH2 groups from lysine into xe2x80x94SH groups. A practical drawback with the maleimide reagents is that they are difficult molecules to obtain synthetically.
The chemical formula of phenyl isothiocyanate, also known as isothiocyanatobenzene or isothiocyanic acid phenyl ester is shown below. 
As noted in the Merck Index, 11th Ed., Susan Budavari, et al., eds., Merck and Co., Inc. (Rahway, N.J.: 1989), p. 7275, phenyl isothiocyanate is known to be used as a derivatizing agent for primary and secondary amines. Such derivatization of primary and secondary amines has typically been used for carrying out Edman degradation and amino acid analyses by HPLC.
This art does not teach the introduction of macromolecules such as polyethylene glycol by the use of a phenylisothiocyanate-containing molecule. Nor are there available any commercial sources of polyethylene glycols or other such macromolecules activated with phenylisothiocyanate. Furthermore, the precursors for obtaining such activated macromolecules are not commercially available or known in the art, e.g. agents containing both a isothiocyanate group and an isocyanate group.
The direct linkage of an alcohol-containing polysaccharide to an amine-containing protein vie reductive amination is known for linking antigenic polysaccharides to carrier proteins for the preparation of vaccines. Aldehyde groups are prepared on either the reducing end [Poren et al. (1985) Mol. Immunol., 22:907-919] or the terminal end [Anderson et al. (1986) J. Immunol., 137:1181-1186] of an alcohol-containing oligosaccharide or relatively small polysaccharide, which are then linked to an amine group in the protein via reductive amination. U.S. Pat. No. 4,356,170 discloses such preparation of useful polysaccharides that are reduced and then oxidized to form compounds having terminal aldehyde groups that can be reductively aminated onto free amine groups of carrier proteins such as tetanus toxoid and diphtheria toxoid with or without significant cross-linking. Several of the problems associated with the attachment of biomolecules to macromolecules are overcome by use of the reagents and processes described hereinafter.
The present invention provides reagents and processes for the linking of alcohol-containing macromolecules, M, to amine-containing biomolecules, B. The compositions of the present invention are linking reagents, linking reagent precursors and reacted linking reagents having (i) an isothiocyanate or a thiourea derivative of an amine-containing biomolecule B, and (ii) one or two other phenyl substituents in the meta- or para-position that is (a) isocyanate, (b) acylazide, or (c) a urethane derivative of an alcohol-containing macromolecule M. Examples of alcohol-containing macromolecules, M, include but are not limited to polysaccharides and hydroxylated silica derivatives. Examples of amine-containing biomolecules, B, include but are not limited to nucleic acids and polypeptides.
A general chemical formula for a linking reagent or linking reagent precursor is shown below having (i) isothiocyanate, and (ii) one or two other phenyl substituents in the meta- or para-position that is (a) isocyanate, (b) acylazide, or (c) a urethane derivative of an alcohol-containing macromolecule M. 
The subscript, n, is 1 or 2, denotes the number of R substituents on the phenyl ring. R is xe2x80x94NHC(O)xe2x80x94Oxe2x80x94M, xe2x80x94NCO or xe2x80x94C(O)N3., with R of xe2x80x94NCO or xe2x80x94C(O)N3 in linking reagent precursors. M is a reacted alcohol-containing macromolecule. The R phenyl substituents are in the meta-, di-meta- or para-positions relative to the isothiocyanate group. The di-meta di-substituted phenylisothiocyanate is preferred over the meta, para-di-substituted phenylisothiocyanate.
A general formula for a reacted linking reagent of the present invention is shown below having (i) a thiourea derivative that is a reaction product of an amine-containing biomolecule, B, and an isothiocyanate, and (ii) one or two other phenyl substituents in the meta- or para-position that is a urethane derivative of an alcohol-containing macromolecule, M. The subscript n is 1 or 2, as before. 
The isothiocyanate moiety of the linking reagent of the invention reacts with a primary amino group of a biomolecule B to be linked using the linking reagent, to form a thiourea moiety. In the balanced chemical reaction, there is effectively no leaving group from the reaction of the isothiocyanato group with the amino group. Thus, in some embodiments, purification issues due to reaction side products and the costs associated therewith are eliminated.
Some embodiments of linking reagent precursors are chemically facile and relatively inexpensive to prepare. Some embodiments of the linking reagent precursors are stable enough for preparation and shipment with a reasonable shelf life.
In some embodiments of the invention, linking reagents are useful for increasing the hydrodynamic volume of biomolecules, which may prolong the half-life of a biomolecule circulating in blood in a living body. In some embodiments, linking reagents are useful for linking a biomolecule to a surface.
The various embodiments of the present invention has several benefits and advantages, however all embodiments do not necessarily provide all of the below-listed benefits and advantages. Further benefits and advantages of embodiments of the invention will be recognized by workers in the art.
One benefit of several embodiments of the invention is that it provides an embodiment of a novel, activated pegylation reagent that permits rapid, efficient, one-step production of pegylated biomolecules having stable, enhanced physiological properties in a predictable and reproducible manner on a meaningful, economical scale.
An advantage of several embodiments of the invention is that it provides an embodiment of a reagent that does not suffer from a significant hydrolysis rate in aqueous reaction media that are buffered at a pH value of about 5.5 to about 8.5, maintains good rates of reactivity, is specific for primary amine groups in target biomolecules and does not produce a leaving group upon covalent attachment to the target biomolecule.
A further benefit of several embodiments of the invention is that it provides an embodiment that permits direct PEG modification of one or more-amino (preferably xe2x80x94NH2) groups or moieties on the biomolecule without first converting those amino groups to one or more sulfhydryl (xe2x80x94SH) groups.
The present invention provides reagents and processes for the linking of alcohol-containing macromolecules, M, to amine-containing biomolecules, B. The compositions of the present invention are linking reagents, linking reagent precursors and reacted linking reagents having (i) an isothiocyanate or a thiourea derivative of an amine-containing biomolecule B, and (ii) one or two other phenyl substituents in the meta- or para-position that is (a) isocyanate, (b) acylazide, or (c) a urethane derivative of an alcohol-containing macromolecule M. Examples of alcohol-containing macromolecules, M, include but are not limited to polysaccharides and hydroxylated silica derivatives. Examples of amine-containing biomolecules, B, include but are not limited to nucleic acids and polypeptides.
A general chemical formula for a linking reagent or linking reagent precursor is shown below having (i) isothiocyanate, and (ii) one or two other phenyl substituents in the meta- or para-position that is (a) isocyanate, (b) acylazide, or (c) a urethane derivative of an alcohol-containing macromolecule M. 
The subscript, n, is 1 or 2, denoting the number of R substituents on the phenyl ring. R is xe2x80x94NHC(O)xe2x80x94Oxe2x80x94M, xe2x80x94NCO or xe2x80x94C(O)N3. M is a reacted alcohol-containing macromolecule. The R phenyl substituents are in the meta-, di-meta- or para-positions relative to the isothiocyanate group.
A general chemical formula for a linking reagent precursor having isothiocyanate and isocyanate moieties is shown below, with n being 1 or 2, as before. 
A general chemical formula for a linking reagent precursor having isothiocyanate and the precursor to the isocyanate, an acyl azide, is shown below, with n being 1 or 2, as before. 
A general formula for a linking reagent that is a kind of activated alcohol-containing macromolecule is shown below, having an isothiocyanate group and xe2x80x94NHC(O)xe2x80x94Oxe2x80x94M, where M is a reacted alcohol-containing macromolecule, with n being 1 or 2, as before. In a preferred embodiment where n is 1, the isothiocyanate moiety is in the 4-position (para) relative to the xe2x80x94NHC(O)xe2x80x94Oxe2x80x94M. 
The isothiocyanate moiety of the linking reagent of the invention reacts with a primary amino group (H2Nxe2x80x94B) to form a thiourea moiety, as shown in Scheme 2 below. In the balanced chemical reaction, there is effectively no leaving group from the reaction of the thiocyanato group with the amino group. The linking reagent can also react with a secondary amine. 
Primary amines present in a typical protein are part of a lysine (Lys or K; amino pK=10.5) amino acid residue, and also the amino terminus of the peptide backbone.
A general formula for a reacted linking reagent of the present invention is shown below having (i) a thiourea derivative that is a reaction product of an amine-containing biomolecule, B, and an isothiocyanate, and (ii) one or two other phenyl substituents in the meta- or para-position that is a urethane derivative of an alcohol-containing macromolecule, M. The subscript n is 1 or 2, as before. 
Also contemplated is an alcohol-containing macromolecule, M, that is derivatized with more than one phenylisothiocyanate group. A general formula shown below illustrates a macromolecule having two phenylisothiocyanate groups. Such a molecule is useful for cross-linking two biomolecules, B, or two amino groups within a biomolecule. 
A preferred form of the reagent for a non-crosslinking pegylation reagent, represented by the formula shown below, is a phenyl isothiocyanate derivative of methoxy-polyethylene glycol (mPEG). 
The methoxy-PEG moiety in the formula above is represented by xe2x80x94(O)xe2x80x94CH2CH2xe2x80x94(OCH2CH2)xxe2x80x94Oxe2x80x94CH3, where x is an average repeat unit number that is about 5 and about 500, preferably about 50 to about 300.
A preferred form of the reagent for a crosslinking pegylation reagent, represented by the formula shown below, is a di-(phenylisothiocyanate) derivative of PEG. 
The PEG moiety in the formula above is represented by xe2x80x94(O)xe2x80x94CH2CH2xe2x80x94(OCH2CH2)xxe2x80x94Oxe2x80x94, where x is an average repeat unit number that is about 5 and about 500, preferably about 50 to about 300.
A preferred form of the reagent for a linking pegylation reagent where n is 2, represented by the formula shown below, is a di-meta pegylated derivative of phenylisothiocyanate. 
The mPEG moiety in the formula above is represented by xe2x80x94(O)xe2x80x94CH2CH2xe2x80x94(OCH2CH2)xxe2x80x94Oxe2x80x94CH3, where x is an average repeat unit number that is about 5 and about 500, preferably about 50 to about 300.
The isothiocyanate moiety of the linking reagent of the invention reacts with a primary amino group of a biomolecule B to be linked using the linking reagent, to form a thiourea moiety. In the balanced chemical reaction, there is effectively no leaving group from the reaction of the isothiocyanato group with the amino group. The linking reagent can also react with a secondary amine. Preferably, for a protein, the primary amine is from the side chain of lysine or the amino terminus.
A contemplated biomolecule may be a polypeptide such as an antibody, enzyme, or protein, or a nucleic acid. Some exemplary polypeptides that benefit from pegylation include, but are not limited to, hemoglobin, bilirubin oxidase and insulin. Several contemplated biomolecules are discussed in Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, J. M. Harris, ed. Plenum, NY, 1992 and Polyethylene Glycol Chemistry and Biological Applications, J. M. Harris and S. Zalipsky, eds., ACS, Washington, 1997. In another embodiment, the invention contemplates the linkage of the isothiocyanate to an amine of a biomolecule that is not a typical peptide residue. The invention contemplates the linking of an amine-containing biomolecule such as a drug or pro-drug, a hapten, a cytokine, a ligand for a receptor, a peptide analog, a nucleic acid base or nucleic acid analog.
In a method for providing a pegylated protein, an amine-containing protein is linked to a large, alcohol-containing PEG macromolecule, such as a PEG-phenyl isothiocyanate compound. Such a pegylated protein provides a protein that can circulate in the blood with a longer half-life than the non-pegylated protein.
In a method for providing an antibody linked to a surface, an amine-containing antibody is linked to an alcohol-containing cellulose derivatized with a phenyl isothiocyanate compound. Such a linked antibody is useful, for example in methods where separation between material that binds to the antibody from material that does not bind to the antibody is desired.
In a method for providing a protein linked to a surface, an amine-containing antibody is linked to an alcohol-containing cellulose derivatized with a phenyl isothiocyanate compound.
In a method for providing a ligand linked to a surface, an amine-containing ligand is linked to an alcohol-containing surface that has been derivatized with a phenyl isothiocyanate. Such a linked ligand is useful, for example in methods where separation between material that binds to the ligand from material that does not bind to the ligand is desired. Such an alcohol-containing surface might be a cellulose membrane or a silica bead having reactive hydroxyl groups.
In a method for providing a multi-subunit protein with enhanced stability, amine-containing proteins are crosslinked intramolecularly with a bifunctional alcohol-containing molecule, such as a PEG di-(phenylisothiocyanate) compound. Such a cross-linked multisubunit protein is useful, for example in studies of the relationships between subunits or to ascertain what proteins are in a complex, such as a transcription complex with effectors.
In an embodiment where n is 1, the R group is preferably in the para position. Thus, in a preferred embodiment where n is 1, the isothiocyanate moiety is in the 4 position (para) relative to the isocyanate (xe2x80x94NCO), xe2x80x94C(O)N3, or xe2x80x94NHC(O)xe2x80x94Oxe2x80x94M moiety.
In an embodiment where n is 2, there are two R substituents on the phenyl ring. The di-meta di-substituted phenylisothiocyanate is preferred over the meta, para-di-substituted phenylisothiocyanate. In an embodiment where n is 2, the invention contemplates R groups that are not identical, such as different M groups in xe2x80x94NHC(O)xe2x80x94Oxe2x80x94M, or an xe2x80x94NCO group and an xe2x80x94NHC(O)xe2x80x94Oxe2x80x94M group. Thus, reaction of less than 100 percent of the xe2x80x94NCO to form xe2x80x94NHC(O)xe2x80x94Oxe2x80x94M is contemplated, as is the use of a mixture of macromolecular forms (e.g. PEG that has a range of chain lengths, as is typical in some commercially available PEG preparations).
The azido compound, where R is xe2x80x94C(O)N3, is stable, and is contemplated for use as a general precursor linking reagent. When R is xe2x80x94NHC(O)xe2x80x94Oxe2x80x94M, M is an alcohol-containing macromolecule is derivatized with one or more, but preferably only one or two, phenylisothiocyanate groups. Where M is derivatized with more than one phenylisothiocyanate group, the reagent is a crosslinking reagent. Where M is PEG, and M is derivatized with more than one phenylisothiocyanate group, the reagent is a crosslinking pegylation reagent.
The invention contemplates the linkage of the isothiocyanate group to an amine, preferably a primary amino group of a biomolecule B to be linked using the linking reagent. Such a biomolecule is preferably a nucleic acid or a polypeptide, e.g. an antibody, enzyme, or protein.
In an embodiment, the invention contemplates the linkage of the isothiocyanate to an amine of a biomolecule that is not a typical peptide residue. The invention contemplates the linking of an amine-containing biomolecule such as a drug or pro-drug, a cytokine, a ligand for a receptor (e.g. example streptavidin), a peptide analog, a nucleic acid base or nucleic acid analog.
In a method for providing a pegylated polypeptide, an amine-containing polypeptide is linked to a large, alcohol-containing PEG macromolecule, such as a PEG-phenyl isothiocyanate compound. Such a pegylated polypeptide provides a polypeptide that can circulate in the blood with a longer half-life than the non-pegylated form. Such a pegylated polypeptide is thus useful in a method of treating a mammal (including homo sapiens) involving the administration of a polypeptide.
The invention contemplates a method of making a stabilized peptide through attachment of a polyethylene glycol to a peptide. In a preferred embodiment, a bifunctional molecule (a phenyl group with an isothiocyanate moiety and an isocyanate moiety) serves to crosslink the polyethylene glycol moiety to a peptide via an amine group, preferably a primary amino group, such as on a lysine side chain.
A polyethylene glycol (PEG) compound can itself be quite varied in composition, but contains at least one poly(oxyethylene) chain [(xe2x80x94CH2CH2Oxe2x80x94)x] having an average molecular weight of about 300 (x is 5) to about 22,000 (x is 500), with an average molecular weight of about 2,300 (x is 50) to about 13,300 (x is 300) being more preferred. More specifically, the reacted PEG compound group M of the linker corresponds to the formula xe2x80x94CH2CH2xe2x80x94(CH2CH2O)nxe2x80x94CH2CH2Y where X, n and R are defined and discussed hereinbelow.
In the above formula, x is a number having an average value of about 5 to about 500, and more preferably about 50 to about 300. It is well known that the higher molecular weight PEG compounds are usually mixtures rather than pure compounds having a single molecular weight. As a result, x, the number of ethyleneoxy repeating units, is a number that is an average number. The terminal Y group is xe2x80x94OH or a C1-C10 hydrocarbyl ether (alkoxy group) having a molecular weight of up to about one-tenth of the xe2x80x94(CH2CH2O)nxe2x80x94 portion.
Exemplary C1-C10 hydrocarbyl ether groups are well known and include alkyl, alkenyl, alkynyl and aromatic ethers. Illustrative C1-C10 ethers thus include methyl, which is most preferred, ethyl, isopropyl, n-butyl, cyclopentyl, octyl, decyl, 2-cyclohexenyl, 3-propenyl, phenyl, 1-naphthyl, 2-naphthyl, benzyl, phenethyl and the like ethers. These ether groups can also be named methoxy, ethoxy, isopropoxy, n-butoxy, cyclopentyloxy, octyloxy, decyloxy, 2-cyclohexenyloxy, 3-propenyloxy, phenoxy, 1-naphthoxy, 2-naphthoxy, enzyloxy and phenethyloxy. A C1-C6 hydrocarbyl group is a particularly preferred Y group.
The molecular weight of a C1-C10 hydrocarbyl ether can be up to about one-tenth of the weight of the xe2x80x94(CH2CH2O)xxe2x80x94 portion of the PEG group. Thus, where x is 20, the xe2x80x94(CH2CH2O)xxe2x80x94 portion has a molecular weight of 880 (20xc3x9744) so that the molecular weight of Y can be up to about 90, or about the weight of a phenoxy group. It is more preferred that the molecular weight of the C1-C10 hydrocarbyl group be about 0.2 to about 2 percent of the molecular weight of the xe2x80x94(CH2CH2O)xxe2x80x94 portion.
The linker molecules of the invention are useful in a variety of methods and assays involving amine-containing peptides or proteins. The invention can be used so that detection enzymes are the amine-containing biomolecule B that is linked to a macromolecule or surface, M, such as cellulose, for use in an assay. Skilled workers in the art can appreciate other methods of using the linker of the invention in their assays with their own amine-containing biomolecules, B, and alchohol-containing macromolecules, M.
For example, streptavidin, such as the wild type or mutants taught in U.S. Pat. No. 6,312,916 granted Nov. 6, 2001, can be useful in binding biotinylated molecules. The streptavidin is linked to a macromolecule, such as PEG, which can change a molecular weight cut off and permit dialysis-type binding assays. The streptavidin B is linked to a macromolecule such as a cellulose membrane, M, which can then be washed with solutions that may contain biotinylated molecules.
Contemplated hydroxy-containing surfaces include, but are not limited to, appropriately derivatized silica, cellulose or gold.
Contemplated hydroxy-containing macromolecules include polysaccharides. On large polysaccharides, one or more of the hydroxy groups may be reacted with a linking reagent precursor. Reaction with a di-meta compound may result in crosslinking of a polysaccharide chain. The carbohydrate itself can be synthesized by methods known in the art, for example by enzymatic glycoprotein synthesis as described by Witte et. al. (1997) J. Am. Chem. Soc., 119:2114-2118.
Several oligosaccharides, synthetic and semi-synthetic, and natural, are discussed in the following paragraphs as examples of oligosaccharides that are contemplated haptens to be used in making a HBc conjugate of the present invention. U.S. Pat. No. 4,220,717 also discloses a polyribosyl ribitol phosphate (PRP) hapten for Haemophilus influenzae type b. Andersson et al., EP-0 126 043-A1, disclose saccharides that can be used in the treatment, prophylaxis or diagnosis of bacterial infections caused by Streptococci pneumoniae. European Patent No. 0 157 899-B1, the disclosures of which are incorporated herein by reference, discloses the isolation of antigenic pneumococcal polysaccharides.
The optimal ratio of macromolecule polysaccharide M to biomolecule B in the linked form depend on the particular polysaccharide, the biomolecule, and the linker molecule used.
In a method for providing an antibody linked to a surface, an amine-containing antibody is linked to an alcohol-containing cellulose derivatized with a phenyl isothiocyanate compound or other hydroxy-containing surface. Such a linked antibody is useful, for example in methods where separation between material that binds to the antibody from material that does not bind to the antibody is desired.
In a method for providing a protein linked to a surface, an amine-containing antibody is linked to an alcohol-containing cellulose derivatized with a phenyl isothiocyanate compound.
In a method for providing a ligand linked to a surface, an amine-containing ligand is linked to an alcohol-containing cellulose derivatized with a phenyl isothiocyanate. Such a linked ligand is useful, for example in methods where separation between material that binds to the ligand from material that does not bind to the ligand is desired.
In a method for providing a multi-subunit protein with enhanced stability, amine-containing proteins are crosslinked intramolecularly with a bifunctional alcohol-containing molecule, such as a PEG di-(phenylisothiocyanate) compound. Such a cross-linked multisubunit protein is useful, for example in studies of the relationships between subunits or to ascertain what proteins are in a complex, such as a transcription complex with effectors. Also contemplated is linking between different molecules in an associated complex, for example transcription factors with RNA polymerase.
A preferred pegylation reagent according to the invention is made using methods known in the art or their equivalents.
In one example of the invention, the preparation of a PEG molecule having a phenyl isothiocyanate activating group is carried out as shown in Scheme 3, below. Briefly, a para-aminobenzoic acid is reacted with thiophosgene to produce 4-carboxyphenyl isothiocyanate, as described in Example 1. The carboxy moiety is activated as the azide to form isothiocyano benzoyl azide by known methods, such as that described in Example 2. The azide-activated carboxy moiety was then heated to cause internal rearrangement to isocyanophenyl isothiocyanate, by the Curtius Rearrangement described in Example 3. 
The invention contemplates lirking reagents having one equivalent of hydroxy-containing macromolecule. A contemplated linking reagent is not limited to a para-substituted phenyl isothiocyanate. Meta- and di-meta-substituted phenyl isothiocyanate compounds are also contemplated. The synthesis of the para-substituted compound is shown below in Examples 1-3, starting with para-aminobenzoic acid. A corresponding meta-substituted aminobenzoic acid compounds are commercially available.
The invention contemplates bifunctional linking reagents having more than one phenyl isothiocyanate groups, which are useful as crosslinking reagents. Using the azide-activated synthetic isocyanate reaction described above, a hydroxy-containing macromolecule is linked to the phenyl isothiocyanate group. The use of a macromolecule with more than one hydroxy group, and appropriate adjustments of stoichiometry and reaction conditions, results in a reagent that has more than one phenyl isothiocyanate group. For example, a bifunctional PEG reagent is made using a PEG diol, as illustrated by Example 4, below. Such bifunctional linking reagents are useful for linking two amine-containing biomolecules (the same or different biomolecules).
The invention contemplates linking reagents having one or more hydroxy-containing macromolecules on a single phenyl isothiocyanate group. Such reagents are made using the analogous procedures to those described herein in detail for the mono-substituted phenyl isothiocyanate compound. For example, a di-meta reagent is made starting with 5-aminoisophthalic acid, commercially available (e.g. Aldrich Product No. 18,627-9). The conversion of the amino group to isothiocyanate then proceeds as described in the Examples below, using CSCl2, NaOAc and H2O, using methods known in the art. A preferred method of making a di-meta reagent is shown in Scheme 4 below. Commercially available amino isophthalic acid serves as the starting material that is converted to the corresponding isothiocyanate compound, 3,5-dicarboxyphenyl isothiocyanate, using thiophosgene, C(S)Cl2, in the presence of an aqueous solution of sodium acetate as illustrated below in Example 5. The two carboxylic acid moieties are then activated with sodium azide in the presence of phenyl dichlorophosphate and pyridine, as illustrated in Example 6 below, to provide the corresponding acylazido phenylisothiocyanate. The acylazido moieties convert smoothly to isocyanate moieties via the Curtius rearrangement, which then react with an alcohol-containing macromolecule, such as PEG, to provide a contemplated di-meta-substituted phenylisothiocyanato reagent. 
The linking of two hydroxy-containing macromolecules to the di-meta-substituted reagent also proceeds by methods known in art, examples of which are provided hereinbelow. Preferably, one hydroxy group from each of two macromolecules reacts with a single di-meta isocyanate-substituted phenyl isothiocyanate compound to form a di-meta linking reagent.
Thus is it recognized that the methods described herein are useful to activate a variety of hydroxy-containing macromolecules, including hydroxy and polyhydroxy compounds. Contemplated examples include but not limited to methoxy PEG, PEG-diols and branched PEGs of various molecular weights. Hydroxy and polyhydroxy compounds other that polyethylene glycols are contemplated, including but not limited to celluloses and starches. Such reagents adapting methods known in the art for reacting phenylisocyanates with hydroxl-containing molecules, such as described herein adapted for the molecules of interest, by adjusting the amount of p-isothiocyanobenzoylazide added to match the correct stoichiometry of macromolecular hydroxyls present. Normally, a stoichiometric or slight excess (zero to ten percent molar excess relative to the hydroxy; where zero percent excess is a one-to-one molar ratio) of the azide is added. For instance, dry, insoluble surfaces, i.e., cellulose bearing a plurality of primary hydroxyl groups is activated by soaking the surface in a solution of p-isothiocyanophenyl isocyanate at 20-60xc2x0 C. (prepared in situ), as described in Example 5, below, or hydroxy-derivatized silica. The activated surface is ready for linking to an amine-containing molecule, useful for a wide variety of applications.
The activated supports thus obtained are useful for immobilizing functional proteins such as enzymes or antibodies under mild conditions (e.g. pH 7.4-8.25 10 mm bicarbonate buffer). Likewise, immunoconjugates of small, hydroxy-containing haptens, e.g. vitamin B-12, hydroxyprogesterone, and digoxigenin, are made utilizing a contemplated isothiocyanophenyl isocyanate. A protocol for linking small, hydroxy-containing molecules to an isocyanate compound is described in M. E. Annunziato, et al., Bioconjugate Chem., 4:212-218 (1993), the disclosures of which are incorporated in full herein by reference.
The phenyl isothiocyanate moiety is stable against hydrolysis in aqueous buffers, and it maintains excellent rates of reaction specifically with primary amines in target biomolecules. Bi- and multi-functional linking reagents are thus possible and practical for the efficient derivatization of target molecules for the purpose of establishing inter and/or intra molecular crosslinks which stabilize native tertiary structure.